Compositions for Enhancing Hyperthermia Therapy

ABSTRACT

A composition for decreasing proliferation of cancer cells undergoing hyperthermia therapy is described. Such compositions can include a combination of selenium and fish oil. The respective quantities of selenium and fish oil are provided in amounts effective to synergistically increase the sensitivity of the cancer cells to temperatures in excess of 37° C.

This application is a continuation of U.S. patent application Ser. No.17/163,419, filed Jan. 30, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/007,524, filed Jun. 13, 2018, which claims thebenefit of U.S. Provisional Application No. 62/519,090 filed on Jun. 13,2017. These and all other referenced extrinsic materials areincorporated herein by reference in their entirety. Where a definitionor use of a term in a reference that is incorporated by reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein is deemed to be controlling.

FIELD OF THE INVENTION

The field of the invention is compositions for use with hyperthermia totreat cancer.

BACKGROUND

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Hyperthermia (i.e., exposure to temperatures that exceed normal bodytemperature) has been used to treat various cancers. In some instanceshyperthermia is used to cause cancer cells to become more susceptible tochemotherapeutic agents or to radiation, and serves as an adjunct tosuch therapies. In other instances hyperthermia can be used to kill ordamage cancer cells outright, however in such applications thetemperatures used risk damage to normal cells.

Hyperthermia can be applied locally, regionally, or to the whole body.Local hyperthermia is frequently used to produce very high temperaturesthat are restricted to a tumor site, resulting in thermal ablation. Thisis typically restricted to localized tumors that are exposed at the bodysurface or are accessible to a thin needle or probe. The size of tumorsthat can be treated in this fashion is also limited (generally to aroundtwo inches or less). Regional hyperthermia provides heat to a particularbody region, such as a limb, organ, or body cavity. This can beaccomplished by isolation perfusion (i.e., heating blood using anexternal device and directed it into the circulatory system supplyingthe region) or through the application of RF or microwave energy. Thetemperatures used for regional hyperthermia are too low to result inkilling of cancer cells alone, so this technique is generally used as anadjunct to chemotherapy and/or radiotherapy.

In whole body hyperthermia the patient's body temperature is elevated tofever levels by application of heat (for example, using heated blanketsor immersion in warm water). Temperatures as high as 107° F. are used.It is theorized that this simulates fever and provides short termactivation of certain immune cells, however whole body hyperthermia iscurrently used as an adjunct to chemotherapy.

Attempts have been made to improve the performance of hyperthermiatherapy through the use of various sensitizers. For example, U.S.Provisional Application 60/290681, to Faulk, describes conjugatingsensitizing compounds (such as chemotherapeutic agents) to transferrinto produce transferring conjugates that tend to localize in cancercells. It is not clear, however, how specific this targeting is, whatdegree of sensitization is achieved, or what side effects are producedby the protein conjugate drug. All publications herein are incorporatedby reference to the same extent as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Another approach is suggested in United States Patent ApplicationPublication No. 2004/0072775, to Sobol and Gjerset. This patentapplication teaches genetic modification of cancer cells to re-establishthe function of mutated genes (specifically, p53) that providesensitivity to various cancer treatments, including hyperthermia. It isnot clear, however, how this selective genetic modification is to beachieved in a clinical setting or to what extent it is effective inincreasing sensitivity of cancer cells to hyperthermia alone.

International Patent Application Publication No. WO 2014/054884, toCheon et al., proposes the use of magnetic nanoparticles in hyperthermiatherapy. Such magnetic nanoparticles are thought to provide both asensitizing effect and a source of heat via the application of a highfrequency magnetic field. The need to localize such particles at thetumor site limits the utility of this approach. In addition, the sideeffects of the introduction of such magnetic nanoparticles (particularlyon a repeated or long-term basis) are not clear.

U.S. Patent Application Publication No. US 12/833207, to Lamb et al.,proposes the use of conductive “buttons” positioned at a locationproximate to a tumor. The conductive buttons can be made from metalssuch as gold, silver, aluminum, copper, or alloys and implemented in avariety of shapes and sizes. As with Cheon et al., the need to localizethe conductive buttons at the tumor site and the targeted application ofheat through the conductive buttons limits the utility of this approach.

In yet another approach, U.S. Patent Publication No. US 5,810,888 toFenn et al., proposes the use of a thermodynamic therapy system using aradiation transmission system to focus radiation to heat a treatmentarea in order to activate thermosensitive drug-containing liposomes.However, this approach does not disclose the specific drugs or adjuvantsdelivered by the thermosensitive drug-containing liposomes.

Thus, there is still a need for a well-tolerated and/or non-toxicsensitizer that is effective in causing the death of cancer cellsthrough the use of hyperthermia without the use of adjunct chemotherapyand/or radiotherapy.

SUMMARY OF THE INVENTION

The inventive subject matter provides compositions and methods in whichselenium, fish oil, and/or selenium in combination with fish oilenhances or potentiates the effects of hyperthermia in reducing theproliferation of tumor cells. In a preferred embodiment the selenium isin the form of selenium yeast, an amino acid derived from seleniumyeast, and/or a peptide derived from selenium yeast.

The inventive subject matter contemplates administering a sensitizerselected from the group consisting of selenium, fish oil, and acombination of selenium and fish oil to a cancer cell. It iscontemplated that the selenium, fish oil, or a combination of seleniumand fish oil are administered in sufficiently high doses depending onthe application to increase the sensitivity of the target cancer cellsto hyperthermia. In preferred embodiments, both selenium and fish oilare administered to advantageously increase the sensitivity of cancercells to thermotherapy more than either fish oil or selenium alone.

One embodiment of the inventive concept is a method of treating cancercells that includes administering fish oil formulated as listed in Table1 and inducing hyperthermia in a patient, where the fish oil is providedin an amount that provides a synergistic effect in reducing cancer cellproliferation. In preferred embodiments, the fish oil is provided to thepatient prior to the initiation of thermotherapy. In other embodiments,the fish oil can be administered concurrently with thermotherapy.

Another embodiment of the inventive concept is a method of treatingcancer cells that includes administering selenium in the form ofselenium yeast formulated as listed in Table 1 and inducing hyperthermiain a patient, where the selenium is provided in an amount that providesa synergistic effect in reducing cancer cell proliferation. In preferredembodiments, the selenium is provided to the patient prior to theinitiation of thermotherapy. In other embodiments, the selenium can beadministered concurrently with thermotherapy.

A preferred embodiment of the inventive concept is a method of treatingcancer cells that includes administering both fish oil and selenium inthe form of selenium yeast formulated as listed in Table 1 and inducinghyperthermia in a patient, where the fish oil and the selenium isprovided in an amount that provides a synergistic effect in reducingcancer cell proliferation. In preferred embodiments, the fish oil andthe selenium are provided to the patient prior to the initiation ofthermotherapy. In other embodiments, the fish oil and selenium can beadministered concurrently with thermotherapy.

It is contemplated that thermotherapy can be administered at anytemperature that increases the temperature of bodily tissue above anormal body temperature that is effective to reduce cancer cellproliferation by allowing better perfusion of cancer cells by oxygen andmedication. However, the inventive contemplates that thermotherapy canbe administered at any temperature or combination of temperatures (e.g.,a variable temperature hyperthermia session) between 37° C. and 44° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting the effects of fish oil on thesensitization of MDA-MB-231 breast cancer cells to hyperthermicconditions.

FIG. 2 is a bar graph depicting the effects of fish oil and selenium,independently and in combination, on the sensitization of HT-29 coloncancer cells to hyperthermia temperatures.

FIG. 3 is a bar graph depicting the effect of fish oil and selenium,independently and in combination, on the sensitization of BFTC-905bladder cancer cells to hyperthermia temperatures.

FIG. 4 is a line graph depicting the effect of selenium, fish oil, andselenium/fish oil combinations of the proliferation of A549 lung cancercells.

FIG. 5 depicts the modulation of pAMPKa and COX-2 concentration byselenium yeast and fish oil.

DETAILED DESCRIPTION

Hyperthermia is commonly induced in cancer-affected tissues, such astumors, in order to reduce, inhibit, or reverse the growth of cancercells. Often, hyperthermia therapy accompanies radiotherapy andchemotherapy in order to maximize efficacy of anti-cancer treatments.However, conventional hyperthermia therapies do not incorporate the useof adjuvants to enhance the efficacy of hyperthermia in reducing theproliferation of cancer cells. Methods and compositions to enhance theanti-cancer effects of hyperthermia therapy using fish oil and selenium,individually or in combination, are disclosed herein. Such selenium canbe in the form of selenium yeast, an amino acid derived from seleniumyeast, and/or a peptide derived from selenium yeast. Approximately, asused herein, is defined as ±5% of a stated value.

As used herein, the fish oil can contain about 220 mg docosahexaenoicacid (DHA) and about 330 mg eicosapentaenoic acid (EPA), which areprecursors of particular eicosanoids that can reduce inflammation in thebody. In preferred embodiments, the fish oil contains between about 110mg and about 330 mg of DHA and between about 160 mg and about 500 mg ofEPA. It is contemplated that DHA and EPA combined preferably does notexceed a total of three grams administered over 24 hours. However, it isalso contemplated that DHA and EPA can be present in any quantityeffective to reduce the proliferation of cancer cells when used inconjunction with thermotherapy.

It is contemplated that the combination of DHA and EPA can includebetween about 8% and about 80% of fish oil content depending on variousfactors, such as the source of the omega-3 fatty acids, the processingof the oil, and the amounts of other ingredients in the oil. Sources ofthe fish oil include “oily” fish. For example, herring, Spanishmackerel, salmon, halibut, tuna, anchovies, and sardines can beconcentrated sources of omega-3 fatty acids. However, it is alsocontemplated that any marine source can serve as a source of fish oilcontaining omega-3 fatty acids. In some embodiments fish oil can besourced from fish with lower concentrations of omega-3 fatty acids intheir tissues, including, for example, cod, flounder, and snapper. Insome embodiments a fish oil equivalent that includes omega-3 fatty acidscan be sourced from marine algae directly instead of from oily fish.Alternatively, fish oil equivalents including EPA and/or DHA andsuitable for use in formulations of the inventive concept can beobtained from non-marine sources. For example, non-marine sources of EPAand DHA can include flaxseeds, chia seeds, hemp seeds, walnuts, andsoybeans.

As used herein, it is contemplated that selenium is administered inconcentrations between about 500 ng/ml and about 1500 ng/ml. Seleniumsalts can be toxic if administered directly. The inventive subjectmatter contemplates sourcing selenium from selenium yeast, which isproduced by cultivating Saccharomyces cerevisiae or another suitableyeast in a selenium-rich media. By cultivating yeast in a selenium-richmedium, selenium can substitute for sulfur in certain amino acids (e.g.methionine, cysteine), thereby providing a nontoxic source of selenium.It is contemplated that selenium from animal sources can be in the formof selenomethionine, selenocysteine, and/or methylselenocysteine as wellas proteins and peptides incorporating such amino acids.

In some embodiments, selenium can also be sourced from plants. Forexample, bioconcentrated selenium can be sourced from plants. In otherembodiments, soluble selenium (e.g., selenate) found in soil can be asource of selenium. In yet other embodiments, selenium can be sourcedfrom ocean water.

Surprisingly, the Inventor has found that the use of fish oil and/orselenium yeast can complement the effects of hyperthermia on cancercells, and can do so in a synergistic (i.e. greater than additive)fashion. FIG. 1 is a bar graph depicting the effects of fish oil on thesensitization of MDA-MB-231 breast cancer (“BC”) cells to hyperthermicconditions. Breast cancer (“BC”) cells were subjected to control (37°C.) or hyperthermia temperatures (39° C., 41° C.). At each temperature,each set of BC cells was (1) exposed to fish oil in concentrations of 0μM (control at 37° C.), 12.5 μM, 25 μM, and 50 μM, (2) held at 37° C.,39° C., or 41° C. for two hours, (3) held in a CO₂ incubator for 72hours, and (4) checked for cell proliferation as a percentage of that ofcontrol BC cells (cells at control temperature and 0 μM fish oilconcentration. The fish oil administered to the non-controlconcentration BC cells cultures contained 220 mg docosahexaenoic acid(DHA) and 330 mg of eicosapentaenoic acid (EPA) per gram.

BC cells held for two hours at temperatures in excess of 37° C. andadministered no fish oil showed marked reductions in cell proliferationsas a percentage of the control BC cell culture. At 39° C., a reductionof at least 25% in the proliferation of BC cells was observed comparedto the control BC cell culture. At 41° C., a further reduction ofapproximately 50% in the proliferation of BC cells was observed comparedto the control BC cell culture.

BC cells held for two hours at all tested temperatures and administered12.5 μM concentrations of fish oil showed an overall reduction in theproliferation of BC cells with mixed results at varying temperatures. At37° C., a reduction of approximately 30% was observed compared to the BCcontrol. At 39° C., a reduction of approximately 60% was observedcompared to the BC control, indicating a synergistic effect. At 41° C.,a reduction of approximately 50% in the proliferation of BC cells wasobserved compared to the BC control.

BC cells held for two hours at all tested temperatures and administered25 μM concentrations of fish oil showed an overall reduction in theproliferation of BC cells with mixed results at varying temperatures. At37° C., a reduction of approximately 60% was observed compared to thecontrol BC cell culture. At 39° C., a reduction of approximately 65% wasobserved compared to the control BC cell culture. At 41° C., a reductionof approximately 65% in the proliferation of BC cells was observedcompared to the control BC cell culture.

BC cells held for two hours at all tested temperatures and administered50 μM concentrations of fish oil showed an overall reduction in theproliferation of BC cells with mixed results at varying temperatures. At37° C., a reduction of more than 75% was observed compared to thecontrol BC cell culture. At 39° C., a reduction of approximately 80% wasobserved compared to the control BC cell culture. At 41° C., a reductionof approximately 85% in the proliferation of BC cells was observedcompared to the control BC cell culture.

The exposure of MDA-MB-231 breast cancer cells to hyperthermiaconditions (e.g. 39° C. or 41° C.) alone results in a moderate decreasein cell proliferation relative to a 37° C. control. As shown, theeffects of hyperthermia are markedly enhanced (in some instances in asynergistic manner) by simultaneous exposure to fish oil in adose-dependent manner. It should be appreciated that the temperaturesutilized are within the range of temperatures that can be producedsafely in a human body by conventional and relatively simple means, suchas immersion in warm water and/or use of heated blankets. It should beappreciated that these temperatures are considerably below the extremesthat can be employed in hyperthermia therapy.

The Applicant believes, without wishing to be bound by theory, that theeffect of fish oil is reducing cell proliferation in cancer cells is dueat least in part to effects particular concentrations of fish oil havewhen combined with elevated temperature on the cell cycle of the cancercells.

FIG. 2 is a bar graph depicting the effects of fish oil and selenium,individually and in combination, on the sensitization of HT-29 coloncancer cells (“HT cells”) to hyperthermia temperatures. The sensitizingeffects of selenium, fish oil, and selenium and fish oil in combinationare evident in HT-29 colon cancer cells exposed to hyperthermiatemperatures.

HT cells were subjected to 37° C. (control) and hyperthermiatemperatures (39° C. and 41° C.). At each temperature, each set of HTcells was either (1) treated with 0.5 μg/ml of selenium, a 25 μMconcentration of fish oil, or both the 0.5 μg/ml of selenium and the 25μM concentration of fish oil, (2) held at 37° C., 39° C., or 41° C. fortwo hours, (3) held in a CO₂ incubator for 72 hours, and (4) checked forcell proliferation as a percentage of a control HT cell culture. Theselenium was administered in the form of selenium yeast. It iscontemplated that the source of selenium is not limited to seleniumyeast and can be administered using any method known in the art. In theabsence of selenium and/or fish oil, incubation at 39° C. reducedproliferation by about 20% relative to control HT cells and incubationat 41° C. reduced proliferation by about 45% relative to control HTcells.

HT cells held for two hours at all tested temperatures and treated witheither 0.5 μg/ml of selenium, a 25 μM fish oil concentration, or boththe 0.5 μg/ml of selenium and 25 μM fish oil showed an overall reductionin the proliferation of HT cells compared to the control HT cellculture.

At 37° C., treatment of the HT cells with 0.5 μg/ml of selenium resultedin a reduction of proliferation by approximately 10% compared to controlHT cells. At 39° C., treatment of the HT cells with 0.5 μg/ml ofselenium resulted in a reduction of proliferation by approximately 20%compared to the control HT cells. At 41° C., treatment of the HT cellswith 0.5 μg/ml of selenium resulted in a reduction of approximately 60%compared to the control HT cell culture.

At 37° C., treatment of the HT cells with a 25 μM concentration of fishoil resulted in a reduction of proliferation by approximately 25%compared to the control HT cells. At 39° C., treatment of the HT cellswith a 25 μM concentration of fish oil resulted in a reduction ofproliferation by approximately 35% compared to the control HT cells. At41° C., treatment of the HT cells with a 25 μM concentration of fish oilresulted in a reduction of proliferation by approximately 60% comparedto the control HT cells.

At 37° C., treatment of the HT cells with a combination of 0.5 μg/ml ofselenium and a 25 μM concentration of fish oil resulted in a reductionof proliferation by approximately 30% compared to the control HT cells.At 39° C., treatment of the HT cells with 0.5 μg/ml of selenium and a 25μM concentration of fish oil resulted in a reduction of proliferation byapproximately 35% compared to the control HT cells, indicating asynergistic effect. At 41° C., exposure to 0.5 μg/ml of selenium and a25 μM concentration resulted in a reduction of proliferation byapproximately 60% compared to the control HT cells.

The results depicted in FIG. 2 show the surprising result of reducingcell proliferation in HT cells when the cells are exposed to both fishoil and selenium. At each successive elevated temperature, the overallcell proliferation decreases. When combined with either a 0.5 μg/ml ofselenium or a 25 μM concentration of fish oil, the cell proliferationdecreases relative to the control, respectively. Surprisingly, thecombination of both a 0.5 μg/ml of selenium and a 25 μM concentration offish oil shows a synergistic effect by reducing cell proliferation morethan either fish oil or selenium alone.

FIG. 3 is a bar graph depicting the effect of fish oil and selenium,individually and in combination, on the sensitization of BFTC-905bladder cancer cells (BFTC cells) to hyperthermia temperatures. Thesensitizing effects of selenium, fish oil, and selenium and fish oil incombination are evident in BFTC cells exposed to hyperthermiatemperatures.

BFTC cells were subject to control (37° C). and hyperthermiatemperatures (39° C. and 41° C.). At each temperature, each set of BFTCcells was either (1) treated with 0.5 μg/ml of selenium, a 12.5 μMconcentration of fish oil, or both the 0.5 μg/ml of selenium and the12.5 μM concentration of fish oil, (2) held at 37° C., 39° C., or 41° C.for two hours, (3) held in a CO₂ incubator for 72 hours, and (4) checkedfor cell proliferation as a percentage of the BFTC control. The seleniumwas administered in the form of selenium yeast. Again, it iscontemplated that the source of selenium is not limited to seleniumyeast and can be administered using any method known in the art.

BFTC cells held for two hours at all tested temperatures and exposed toeither 0.5 μg/ml of selenium, a 12.5 μM fish oil concentration, or boththe 0.5 μg/ml of selenium and the 12.5 μM concentration of fish oilshowed an overall reduction in the proliferation of BFTC cells comparedto the BFTC control. In the absence of selenium and/or fish oil,incubation at 39° C. reduced proliferation by about 13% relative tocontrol BFTC cells and incubation at 41° C. reduced proliferation byabout 28% relative to control BFTC cells.

At 37° C., treatment of the BFTC cells with 0.5 μg/ml of seleniumresulted in a reduction of proliferation by approximately 10% comparedto the BFTC control. At 39° C., treatment of the BFTC cells with 0.5μg/ml of selenium resulted in a reduction of proliferation byapproximately 15% compared to the BFTC control. At 41° C., treatment ofthe BFTC cells with 0.5 μg/ml of selenium resulted in a reduction ofproliferation by approximately 30% compared to the BFTC control.

At 37° C., treatment of the BFTC cells with a 12.5 μM concentration offish oil resulted in a reduction of proliferation by approximately 30%compared to the BFTC control. At 39° C., treatment of the BFTC cellswith a 12.5 μM concentration of fish oil resulted in a reduction ofproliferation by approximately 35% compared to the BFTC control. At 41°C., treatment of the BFTC cells with a 12.5 μM concentration of fish oilresulted in a reduction of proliferation by approximately 45% comparedto the BFTC control.

At 37° C., treatment of the BFTC cells with 0.5 μg/ml of selenium and a12.5 μM concentration of fish oil resulted in a reduction ofproliferation by approximately 15% compared to the BFTC control. At 39°C., treatment of the BFTC cells with 0.5 μg/ml of selenium and a 12.5 μMconcentration of fish oil resulted in a reduction of proliferation byapproximately 35% compared to the BFTC control. At 41° C., exposure to0.5 μg/ml of selenium and a 12.5 μM concentration resulted in areduction of proliferation by approximately 50% compared to the BFTCcontrol.

The results depicted in FIG. 3 show the surprising result of reducingcell proliferation in BFTC cells when the cells are exposed to both fishoil and selenium. At each successive elevated temperature, the overallcell proliferation decreased. When combined with either a 0.5 μg/ml ofselenium or a 25 μM concentration of fish oil, the cell proliferationdecreases relative to the control, respectively. With the exception ofthe BFTC cell culture treated with 0.5 μg/ml of selenium and a 12.5 μMconcentration of fish oil at 37° C., the mixture of selenium and fishoil shows a synergistic effect by reducing cell proliferation more thaneither fish oil or selenium alone.

FIG. 4 is a line graph depicting the effect of selenium, fish oil, andselenium/fish oil combinations of the proliferation of A549 lung cancercells (A549 cells).

A549 cells were exposed to either 0 μM (PBS), 25 μM, 50 μM, or 100 μMfish oils. At each concentration of fish oil, each set of A5 cells was(1) exposed to either 0 μg/ml of selenium, 0.5 m/ml of selenium, 1 μg/mlof selenium, 2 μg/ml of selenium, or 4 μg/ml of selenium, (2) held at37° C., 39° C., or 41° C. for two hours, (3) held in a CO₂ incubator for72 hours, and (4) checked for cell proliferation as a percentage of PBS.The selenium was administered in the form of selenium yeast. Again, itis contemplated that the source of selenium is not limited to seleniumyeast and can be administered using any method known in the art.

Each set of A549 cells held for two hours at higher concentrations offish oil than PBS resulted in an overall reduction in the proliferationof A549 cells compared to PBS. Additionally, the treatment of cellcultures with increasing concentrations (i.e., from 0.5-4 μg/ml) ofselenium shows the effects on cell proliferation relative to control(PBS only) A549 cells as selenium concentration is increased, inaddition to the effects of the fish oil on the A549 cells. Synergisticeffects are particularly notable at high fish oil concentrations, wherethe addition of even low concentrations of selenium results in aprofound decrease in cell proliferation relative to that of fish oil orselenium (either individually or additively).

At 4 μg/ml of selenium, cell proliferation of each of the A549 cellcultures reduced to approximately 20% of PBS. Between 0 and 4 μg/ml ofselenium, however, exposure to fish oil and selenium, each in increasingamounts, demonstrated an anti-proliferative effect with higher fish oiland selenium concentrations reducing cell proliferation more than lowerfish oil and selenium concentrations.

Table 1 shows the effects of fish oil and selenium on cell cycledistribution of MDA-MB breast cancer cells (MDA-MB cells) at 39° C. and41° C.

TABLE 1 subG1 G0/G1 S G2/M Control (39° C.) 2.1% 66.24% 16.2% 17.55%Control (41° C.) 4.8% 55.91% 22.07% 22.01% Selenium 1000 13.9% 59.15%24.36% 16.5% ng/ml (41° C.) Fish oil 25 μM 13.3% 52.55% 22.37% 20.08%(41° C.) Selenium 1000 28.94% 56.78% 22.13% 21.09% ng/ml + Fish oil 25μM (41° C.)

The concentration of fish oil represents its DHA content. Each gram offish oil contains 220 mg DHA and 330 mg EPA. Cells were incubated athyperthermia temperatures for 2 hours and then placed in a CO2 incubatorfor 72 hours before a cell cycle analysis was performed. Selenium wasadministered in the form of selenium yeast. As shown, the use ofselenium, fish oil, and selenium and fish oil in combination leads to asignificant redistribution of cell cycle occupancy of these cells underhyperthermia condition. Specifically, the percentage of cells in subG1phase (which is associated with apoptosis) is increased.

In addition to modulating cell cycle occupancy, selenium, fish oil, andselenium and fish oil in combination can modulate the concentration ofcertain proteins in cancer cells.

FIG. 5 depicts the modulation of pAMPKa and COX-2 concentration byselenium yeast and fish oil. Increased AMPK signaling is thought toprevent proliferation and metastasis in tumor cells. COX-2 is thought tomodulate cell proliferation and apoptosis in solid tumors, with COX-2inhibition being investigated as a therapeutic mode. Surprisingly, theInventor found that selenium, fish oil, and selenium/fish oilcombinations result in increased levels of pAMPKa and reduced levels ofCOX-2 in A549 lung cancer cells (GAPDH is included as a control). Sucheffects may contribute to the enhancement and/or synergistic effectsseen when these are used in combination with hyperthermia.

Suitable formulations that incorporate fish oil and selenium yeastinclude the nutritional supplement formulation provided in Table 2,which incorporates fish oil and selenium yeast components along withother nutritional components. This nutritional supplement has been foundto have a high level of acceptance and to have unanticipated beneficialanti-tumor activity in combination with conventional therapies. As such,the Applicant believes that use of such a nutritional supplement canprovide a beneficial enhancement of the hyperthermia therapy, and can doso at lower, relatively safe temperatures that are readily achievableusing conventional approaches and safer for patient use. The provisionof such a safety margin in regards to body and local temperature canlead to broader acceptance and use of this non-toxic therapeutic mode.

TABLE 2 Minimum Maximum Unit Component Maltodextrin 10000 50000 mg WheyProtein Isolate 5000 60000 mg Whey Protein Concentrate 1000 50000 mgFructooligosaccharides/Inulin 40 15000 mg Granulated Honey 1000 9000 mgOat Fiber 500 15000 mg Natural French Vanilla Flavor 500 20000 mg SoyProtein 500 50000 mg Brownulated Powdered Brown Sugar 500 10000 mgNatural Vanilla Masking Flavor 500 5000 mg Lecithin 200 10000 mg Milk,Non-fat 50 5000 mg Rice Protein Powder 50 5000 mg Calcium Caseinate 502000 mg Oils Flax Seed Oil 100 7000 mg Canola Oil 100 7000 mg Borage Oil100 7000 mg Olive Oil 100 7000 mg Fish Oil 150 10,000 mg Pure Lemon Oil100 1000 mg Pure Orange Oil 50 1000 mg Mixed Tocopherols 0.5 200 mgVitamins/Minerals Potassium Phosphate 200 1500 mg Calcium Carbonate 1005000 mg Choline Bitartrate 150 2500 mg Sodium Chloride 100 2000 mgCalcium Phosphate Tribasic 100 2000 mg Ascorbic Acid 50 3000 mgPotassium Chloride 50 2000 mg Magnesium Oxide 50 500 mg Selenium Yeast30 4000 mcg Chromium Yeast 30 3000 mcg Molybdenum Yeast 30 2000 mcgInositol 10 5000 mg Zinc Sulfate Monohydrate 5 200 mg Dry Vitamin EAcetate 5 2000 IU Niacinamide 5 500 mg Ferric Orthophosphate 3 100 mgCalcium Pantothenate 3 200 mg Manganese Sulfate Monohydrate 3 100 mgBeta Carotene 1 100 mg Copper Gluconate 1 15 mg Vitamin D3 25 5000 IUVitamin K2 2 1000 mcg Pyridoxine HCl 0.5 200 mg Potassium Iodide 0.51500 mg Riboflavin 0.5 1000 mg Thiamine Hydrochloride 0.5 2500 mg DryVitamin K1 1 500 mcg Vitamin A Acetate 500 100000 IU Folic Acid 10010000 mcg d-Biotin 10 10000 mcg Vitamin B12 1 3000 mcg Amino AcidsL-Carnitine 300 30000 mg L-Glutamine 500 60000 mg L-Arginine Base 50030000 mg Taurine 50 2000 mg L-Lysine 50 2000 mg Alpha Lipoic Acid 101000 mg Resveratrol 15 1500 mg Co-Enzyme Q10 10 5000 mg Glycine 5 1000mg Proline 5 1000 mg Bacterial Cultures Lact. Acidophilus (app. 10billion total) 2 500 mg Bifido Bifidium (app. 10 billion total) 2 500 mgLac. Bulgaricus (app. 10 billion total) 2 500 mg Bifido Longum (app. 10billion total) 2 500 mg Strep. Thermophilus (app. 10 billion total) 2500 mg Enzymes Papain 5 100 mg Pepsin 5 100 mg Lipase 5 100 mg Bromelain5 100 mg Pancreatin 4X 0.5 100 mg Lactase 1 100 mg Betaine HCl 3 100 mgPlant Products Pineapple Juice Powder 2 500 mg Papaya Fruit Powder 2 500mg Quercetin 30 3000 mg EGCG 25 600 mg OPC 15 500 mg Anthocyanins 155000 mg Ellagic Acid 10 300 mg Astaxanthin 2 90 mg Fucoidan 20 1500 mgMushroom Preparation Cordyceps 5 6000 mg Ganoderma Lucidum 15 10000 mgShiitake 40 15000 mg Maitake 30 15000 mg Turkey Tail 30 15000 mg

The composition shown in Table 2 includes components that have variousphysiological and biochemical effects, including anti-inflammatoryactivity, lowering of blood glucose levels, lowering of cholesterol, andanti-tumor activity. Other components provide supplementation ofnecessary vitamins, minerals, and amino acids at elevated levels. Othercomponents (e.g. enzymes, lecithin) serve to aid in digestion andabsorption of components of the composition. The combination provides asynergistic effect that exceeds the simple additive effect of individualcomponents. It should be appreciated that the composition shown in Table2 also includes certain flavorants (e.g. brown sugar, honey, vanillaflavor and masking agent) that serve to improve palatability andacceptance. Certain components (e.g. honey, brown sugar, milk, riceprotein, casein) can provide both flavor and caloric energy. TheInventor has found that the combination of flavorants described above iseffective in providing compliance with consumption of the nutritionalsupplement in effective amounts. In some embodiments, such flavorantscan be excluded without negatively impacting the effectiveness of thenutritional supplement, thereby providing a functional nutritionalsupplement that includes only essential components.

It should be appreciated that components of a nutritional supplement ofthe inventive concept can be provided as powders, granules, liquids,suspensions, and/or emulsions. In a preferred embodiment, components ofthe nutritional supplement are provided as powders and/or granules.Similarly, in preferred embodiments of the inventive concepts componentsof the nutritional supplement are provided in relative amounts asindicated in Table 2. In some embodiments the components of thenutritional supplement are provided as a single, mixed formulation. Inother embodiments components of the nutritional supplement can beprovided as a kit or similar assembly containing different components ofthe formulation segregated or packaged separately (for example, toprovide different storage conditions conducive to component stability).

Components shown in Table 2 can be provided as a single formulation (forexample, as a pill, tablet, capsule, powder, liquid, suspension, etc.)or can be segregated into different formulations (for example, as pills,tablets, capsules, powders, liquids, suspensions, or combinationsthereof). The amounts shown in Table 2 are exemplary, and representtypical daily dosages provided to an adult of normal stature andotherwise normal health. These amounts can be adjusted to account fordifferences in body mass, gender, medical condition, etc. For example, arelatively small patient weighing 40 kilos or less may receive benefitfrom dosages provided at or below the low end of the ranges provided,whereas a relatively large patient weighing 100 kilograms or more mayrequire dosages provided at the high end of the ranges noted (or more).In some embodiments such a daily dose can be distributed as multipledoses throughout the day. In some of such embodiments the composition ofeach of such distributed doses can be identical. In other embodimentsthe composition of such distributed doses can be different, provided thesummation of such doses provides the required supplementation.

It should be appreciated that oils found in the formulation (e.g. FlaxSeed Oil, Canola Oil, Borage Oil, Olive Oil, Fish Oil, Pure Lemon Oil,Pure Orange Oil, Mixed Tocopherols) are at least consumer grade, andpreferably highly purified (>95% pure). It should also be appreciatedthat mineral components (e.g. potassium, calcium, sodium, magnesiumiron, manganese) can be provided as any safe and absorbable salt (e.g. ahalide salt, phosphate salt, carbonate salt, sulfate salt), oxide, ororganic complex (e.g. gluconate). It should also be appreciated thatcertain metals (e.g. chromium, molybdenum, selenium) are supplied in theform of a yeast component, which can include provision as ayeast-containing powder or suspension and/or as a complex with a peptideor amino acid as a result of metabolism of such metals by yeast.Similarly, it should be appreciated that preparation of variousnon-yeast fungi (e.g. Cordyceps, Ganoderma Lucidum, Shiitake, Maitake,Turkey Tail) can include powdered or granular preparation derived fromdried/lyophilized fruiting bodies of such fungi.

A nutritional supplement of the inventive concept can be provided inamounts ranging from about 1 mg/kg body weight to about 100 g/kg body asa unit dose. Such a unit dose can be provided on a schedule ranging from4 times a day to one time per week. The nutritional supplement can beprovided as one or more pills or capsules. Alternatively the nutritionalsupplement can be provided as a powder, granular, and/or liquidformulation that is added to a food or a beverage prior to consumption.In some embodiments the nutritional supplement can be provided as a fooditem, such as a food or candy bar. In other embodiments the nutritionalsupplement can be provided as a solution, suspension, or beverage thatis suitable for oral consumption and/or provision by tube feeding.

It should be appreciated that packaging that excludes light, moisture,and/or oxygen can be used to extend the shelf life of the nutritionalsupplement. Similarly, a nutritional supplement of the inventive conceptcan be packaged with a hygroscopic agent (such as silica gel), anon-reactive gas (such as N2 or a noble gas), and/or under vacuum inorder to extend shelf life. Such packaging can, for example, provide anutritional supplement of the inventive concept in single unit doses andadditionally provide directions for preparation and/or dosing frequency.

The present invention contemplates using hyperthermia in conjunctionwith nutritional supplements with varying concentrations of fish oil andselenium. Hyperthermia regimes consist of heating body tissue tosupra-normal body temperatures to sensitize cancer cells to treatmentmethods or to directly kill cancer cells. For example, hyperthermiaregimes can be administered for 45-60 minutes over 4-12 sessions.Hyperthermia can be applied locally, regionally, or to the whole bodyand sourced from microwave energy, radiofrequency energy, ultrasoundenergy, or any other source of energy sufficient to heat tissue tosupra-normal temperatures. In some embodiment, heat can be directlyapplied to the cancerous tissue, including, for example, by inserting aheated probe inside a tumor.

In one embodiment, a nutritional supplement containing a 25 μMconcentration of fish oil is administered to a patient to be ingestedorally. After waiting a sufficient amount of time to allow the fish oilto be absorbed into the blood stream (e.g., over a period of two hours),heat is applied to the cancerous tissue to raise the temperature of thetissue to supra-normal levels for a 45 minute duration. For example, amicrowave heating element can be applied locally to the surface of theskin of the patient above a tumor. In another example, a probe can beinserted into a tumor and heated. In yet another example, a patient canbe instructed to wear a whole body suit and the patient's body can beheated to 39° C.

In another embodiment, a nutritional supplement containing 1000 ng/ml ofselenium in the form of selenium yeast can be administered to a patientto be ingested orally. After waiting a sufficient amount of time toallow the selenium to be absorbed into the blood stream (e.g., over aperiod of two hours), heat is applied to the cancerous tissue to raisethe temperature of the tissue to supra-normal levels for a 60 minuteduration. As in the preceding embodiment, a microwave heating elementcan be applied locally to the surface of the skin of the patient above atumor. In another example, a probe can be inserted into a tumor andheated. In yet another example, a patient can be instructed to wear awhole body suit and the patient's body can be heated to 39° C.

In a preferred embodiment, a nutritional supplement containing a 25 μMconcentration of fish oil and 1000 ng/ml of selenium in the form ofselenium yeast can be administered to a patient to be ingested orally.After waiting a sufficient amount of time to allow the fish oil and theselenium to be absorbed into the blood stream (e.g., over a period oftwo hours), heat is applied to the cancerous tissue to raise thetemperature of the tissue to supra-normal levels for a 55 minuteduration. As in the preceding embodiment, a microwave heating elementcan be applied locally to the surface of the skin of the patient above atumor. In another example, a probe can be inserted into a tumor andheated. In yet another example, a patient can be instructed to wear awhole body suit and the patient's body can be heated to 39° C.

It is contemplated that the fish oil and selenium individually or incombination and in any respective concentrations can be administeredusing any method effective to expose the cancer cells to the fish oiland/or the selenium. For example, fish oil and selenium can be directlyinjected into the cancerous tissue. In another example, fish oil andselenium can be applied topically to the skin or to the outer mosttissue of an organ to be absorbed into cancerous tissue.

It is also contemplated that heat can be applied to the cancerous tissueusing any method known in the art. For example, a heated element can bedirectly applied to the cancerous tissue or tissue surrounding thecancerous tissue to transfer heat through conduction. In anotherexample, a heating element giving of electromagnetic energy waves can beused to transfer heat including, for example, an infrared heatingelement.

One should appreciate that the disclosed techniques provide manyadvantageous technical effects including sensitizing tumor cells to theeffects of hyperthermia using well-tolerated substances and without theuse of radiation and/or chemotherapeutic agents.

The description of the inventive subject matter herein includesinformation that may be useful in understanding the present invention.It is not an admission that any of the information provided herein isprior art or relevant to the presently claimed invention, or that anypublication specifically or implicitly referenced is prior art.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The detailed description provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A nutritional supplement for decreasingproliferation of a cancer cell being treated with a hyperthermia therapyprotocol, comprising: an effective amount of fish oil; and an effectiveamount of selenium, wherein the effective amount of fish oil and theeffective amount of fish provide a combined synergistic effect indecreasing proliferation of the cancer cell being treated with thehyperthermia therapy protocol.
 2. The nutritional supplement of claim 1,wherein the hyperthermia therapy protocol comprises a temperature of 39°C. to 41° C.
 3. The nutritional supplement of claim 1, wherein theeffective amount of selenium provides 0.5 μg/mL of selenium to thecancer cell.
 4. The nutritional supplement of claim 1, wherein theeffective amount of fish oil provides 12.5 μM To 25 μM fish oil to thecancer cell.